These phenotypes may not all be attributable to altered responses to Wnt proteins

ess, as indicated in wound healing and invasion chamber assay. ONX-0914 web Overall, these data support miR-21 as an essential target of resveratrol for mediating the growth and invasiveness of PC-3MMM2 cells in vitro. Inhibition of miR-21 Expression by Resveratrol Involves Akt Previous studies have proposed that Akt is a direct regulator of miR-21 expression. We therefore examined whether this signaling protein is a target of resveratrol. PC-3M-MM2 cells exhibit high pAkt levels, which were inhibited by resveratrol in a dose-dependent manner. Approximately,52% inhibition was evident at 25 mM resveratrol and,73% inhibition observed at 100 mM resveratrol. The response of 100 mM resveratrol, which appeared to represent a maximum for this compound, was less than LY294002, a known inhibitor of Akt activity. Like resveratrol, LY294002 inhibited miR-21 expression and increased the levels of PDCD4 and PDCD4 luciferase activity. However, LY294002 did not produce any significant change in the levels of maspin. These results support the conclusion that inhibition of Akt by resveratrol could contribute to its suppression of miR-21 expression. While the induction of PDCD4 is consistent with a decrease in miR-21 levels, it is unclear why the levels of maspin were not regulated. Additional experiments were performed in LNCaP and DU145 cells to determine whether resveratrol inhibits pAKT levels in these cells. Resveratrol significantly inhibited pAkt levels in these prostate cancer cell lines without affecting the levels of Akt. Similar responses were observed with LY294002. Overall, these studies suggest that inhibition of Akt phosphorylation could serve as a reasonable mechanism for reducing miR-21 levels and increasing the expression of PDCD4. Additional experiments were performed to test whether the functional responses of resveratrol could be explained by inhibition of the Akt pathway. LY294002 reduced the viability and increased Resveratrol and MicroRNA-21 the apoptosis of PC-3M-MM2 cells, as determined from MTS assay, and Annexin V-FITC and PI staining and TUNEL assay, respectively. In addition, LY294002 inhibited PC-3M-MM2 cell invasiveness. Wound closures in vehicle-treated cells averaged,43%, while they averaged,15% in cultures treated with LY294002. Similarly, LY294002 significantly reduced cell invasion through matrigel. Taken together, these data suggest that Akt plays an essential role in mediating the viability and invasiveness of PC-3M-MM2 cells and could represent a viable target for inhibition by resveratrol. Inhibition of Prostate Cancer Growth and Metastasis by Resveratrol is Associated with Decreased miR-21 Expression In order to determine the in vivo efficacy of resveratrol against prostate cancer, we tested this drug in xenograft model of prostate cancer in SCID mice. These mice were administered vehicle or resveratrol by oral gavage every other day starting 1 week prior to subcutaneous injections of PC-3M-MM2 tumor cells into their flanks. Palpable tumors were detected by treatment day 28 and tumor volumes were measured over an additional 11 days in both groups. Tumor growth increased exponentially in the vehicletreated mice, but the growth in resveratrol-treated mice was significantly reduced beyond day 30. Mean tumor volumes were reduced by,50% in the resveratrol-treated group. Tagging of the PC-3M-MM2 cells with the luciferase gene allowed for whole animal imaging in a Xenogen imager, upon administration of luciferin. Representative imag